Development of Nucleic Acid Lateral Flow Immunoassay Detection Method for Foot-and-mouth Disease Virus

In this article, through the combination of nucleic acid probes and immune chromatography, a simple, sensitive and specific detection system——nucleic acid lateral flow immunoassay (NALFIA) for amplifing foot-and-mouth disease virus (FMDV) 3D RT-PCR products was established.An ultrasensitive nucleic acid biosensor (NAB) based on streptavidin-labeled gold nanoparticles dual labels and lateral flow strip biosensor (LFSB) were used in this system.The biotinylated goat anti-rabbit IgG was marked to the NC membrane as the alleged strip and the anti-digoxin antibody was labeled to the NC membrane to capture the digoxin probe.After assemblying gold-labeled strip and detecting RT-PCR products, the detection limit of NALFIA was 0.3×10^-3 to 3×10^-3 μg/μL.The NALFIA was compared with agar gel electrophoresis analysis, the results showed that the sensitivity of NALFIA was higher than agar gel electrophoresis.There was an excellent agreement between the two methods.NALFIA was a method with high sensitive, low cost and short time.In conclusion, this method provided a good alternative to detect FMDV.     

马铃薯种植机播种作业适应性研究与探索

马铃薯播种机已在甘肃省张掖市马铃薯种植区广泛使用,为了解决不同土壤、不同区域种植模式适应性问题,掌握播种质量存在的问题,为改进马铃薯种植机提供     

Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

To establish a rapid,sensitive and specific assay for the differential detection of Nipah virus (NiV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV),a duplex Real-time RT-PCR was developed with specific primers and probes targeting to the special sequences of NiV M gene and HP-PRRSV nsp2 gene by optimization of reaction conditions.The performance of the assay was linear ranging from 4.6×10¹ to 4.6×10⁷ copies/μL for RNA standard control of NiV M (NiV-M-RNA) and from 4.1×10¹ to 4.1×10⁸ copies/μL for RNA standard control of HP-PRRSV nsp2 (HP-PRRSV-nsp2-RNA),and detection limits of the assay was 46 copies for the NiV-M-RNA and 4.1 copies for the HP-PRRSV-nsp2-RNA,respectively.The coefficients of variation (CVs) of both inter-assay and intra-assay repeatability were less than 2.0%,showing good repeatability.The assay was able to specifically detect NiV and HP-PRRSV simultaneously without cross-reaction with classical swine fever virus (CSFV),porcine epidemic diarrhea virus (PEDV),swine influenza virus (SIV),porcine parvovirus (PPV),pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2).Of the 236 samples from pigs for both NiV and HP-PRRSV detection by the established assay,all the samples were negative for NiV,8 samples were HP-PRRSV positive.In conclusion,this assay offers a useful approach for the differential detection of NiV and HP-PRRSV in clinical specimens from the pigs.     

薄壳山核桃疮痂病菌TaqMan荧光定量PCR检测方法的建立及应用

疮痂病是薄壳山核桃上最具毁灭性的病害,带菌植物材料是传播疮痂病的重要来源。准确、灵敏、快速的检测方法可为该病害流行规律调查和防控提供有力的依据。本文通过比较薄壳山核桃疮痂病菌Venturia effusa及其近似种之间的ITS序列差异,设计了特异性引物和TaqMan探针,建立了薄壳山核桃疮痂病菌的荧光定量PCR检测方法。特异性检测结果表明,该方法可以检测不同地区的薄壳山核桃疮痂病菌菌株,而对其近似种以及薄壳山核桃上的其他真菌均没有信号。本研究建立的检测方法对薄壳山核桃疮痂病菌DNA的最低检测限可达0.5 pg/μL。该方法用于田间样品检测时,检测时间仅需1 h,远快于常规的分离培养法。本研究建立的基于TaqMan探针的荧光定量PCR检测方法为薄壳山核桃疮痂病菌的快速检测和监测提供了有力工具。     

微喷灌正方形布置时喷头组合间距的确定

本文根据微喷灌系统全湿润喷洒灌溉的试验数据和生产考核,分析了微喷灌为正方形布置时,在相应的组合间距下,达到的均匀度指标。SWP-J,SWP-2折射式微喷头正方形布置时,建议其组合间距a×b采用0.7～0.8R,DLXD1.5离心式微喷头组合间距a×b=0.8～0.9R(风速在0～3.8m/s范围),此时喷洒均匀系数不低于0.85。     

垄膜沟种涌泉灌溉技术要素的试验研究

在垄膜沟种农田进行了涌泉灌灌水试验，通过对水流推进过程观测，分析了田面水流运动特性，研究了沟宽对田间灌水均匀度的影响，并对垄膜沟种涌泉灌溉技术要素进行了探讨。结果表明，涌泉灌田面水流推进曲线可用幂函数表示。随着沟宽的增大，灌水均匀度呈下降趋势。从灌水质量和管网成本两方面综合考虑，试验条件下灌水技术要素的入沟流量、沟宽和灌水器间距分别以80～100L/h、20-30cm和4～5m为宜。     

基于双圆盘覆土的种肥分施装置设计与试验

【目的】针对免耕条件下玉米播种施肥时,采用自然回土方式存在的种肥间距不稳定、种子覆土量不足等问题,采用主动覆土方法,设计一种基于双圆盘覆土的种肥分施装置。【方法】设计基于双圆盘覆土的种肥分施装置,利用EDEM对该装置的作业性能进行离散元仿真,研究圆盘直径、圆盘入土深度、圆盘张角、排种管前后间距及圆盘后方开口间距对种肥间距和种子覆土厚度的影响,并在此基础上选取圆盘直径(150,175,200,225,250 mm)、排种管前后间距(60,80,100,120,140 mm)及圆盘后方开口间距(110,135,160, 185,210 mm)为试验因素,利用3因素5水平二次正交回归试验,分析各关键因素对种子覆土厚度的影响。与不加覆土圆盘的装置进行田间对比试验,对基于双圆盘覆土的种肥分施装置作业效果进行验证。【结果】利用Design-Expert软件得出了影响种子覆土厚度的主次因素:圆盘后方开口间距、排种管前后间距、圆盘直径;最优组合是圆盘直径201 mm,圆盘后方开口间距103 mm,排种管前后间距115 mm。田间试验表明,设计的装置种肥间距与种子覆土厚度与仿真优化结果的相对误差分别为7.63%和11.45%;设计的装置种肥间距和种子覆土厚度较不加覆土圆盘的装置分别增加了14.30%和19.30%,种肥间距与种子覆土厚度的变异系数分别为5.21%和7.26%。【结论】所设计的基于双圆盘覆土的种肥分施装置作业效果满足种肥分施要求,为后续种肥分施技术与装置的研发提供了依据。     

非洲猪瘟病毒锁核酸探针荧光定量PCR检测方法的建立

根据非洲猪瘟病毒(African swine fever virus,ASFV)P72基因核苷酸序列设计特异性引物和锁核酸(locked nucleic acid,LNA)-TaqMan探针,建立了基于P72基因的LNA-TaqMan探针的ASFV荧光定量PCR方法.结果显示,所建立的LNA-TaqMan探针荧光定量P...     

猪瘟病毒与非洲猪瘟病毒双重荧光定量PCR方法的建立与应用

为了建立能够猪瘟病毒(Classical swine fever virus, CSFV)和非洲猪瘟病毒(African swine fever virus, ASFV)双重荧光定量PCR方法,试验比对了CSFV与ASFV的基因组序列,针对CSFV 5′-UTR和ASFV B646L保守靶序列,分别设计2条特异性引物及1条TaqMan探针,通过优化扩增体系和程序,建立可同时检测CSFV和ASFV的双重荧光定量PCR方法,并建立该方法的标准曲线,同时用敏感性试验、特异性试验和重复性试验对该方法进行评价,最后检验该方法的应用效果。结果表明：CSFV和ASFV标准曲线的相关系数(R²)分别为0.999和1,线性关系良好;CSFV与ASFV的最低检测浓度分别为1.3×10⁰ copies/μL和2.4×10⁰ copies/μL;与其他常见猪只病原检测无交叉反应;批间与批内变异系数均小于2%,稳定性良好。试验建立的方法对44份临床样本的检测结果与《猪瘟病毒实时荧光RT-PCR检测方法》(GB/T 27540—2011)和《非洲猪...     

二氧化硅生物荧光纳米颗粒的制备与应用

以荧光染料(联吡啶钌配合物)为核,二氧化硅为外壳制备了荧光纳米颗粒.电镜检测结果显示,合成的二氧化硅纳米颗粒粒径为(20±3)nm,分布均匀,形态规则.对荧光纳米颗粒进行生物修饰得到荧光探针,以DNA碱基配对原则为基础建立检测DNA的方法,利用激光扫描共聚焦显微镜研究玻片上的荧光信号和荧光强度.结果表明,该方法不仅可定量检测到4.4×10-8 mol/L的目标DNA3,而且可定性检测目标DNA3、单碱基错配DNA4及随机序列DNA5,为发展DNA检测技术提供了新的思路.